rabbit polyclonal cdk5rap2  (Millipore)

Journal: Nature Cell Biology

Article Title: CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

doi: 10.1038/s41556-024-01366-2

Figure Lengend Snippet: a , Left: representative images of single molecules of indicated purified proteins absorbed on coverslips. Middle: a histogram of single-molecule fluorescence intensities. The numbers of analysed molecules are as follows: n = 21,349 (GFP), n = 28,776 (GFP–EB3) and n = 24,216 (GCP3–GFP), from three independent experiments. Right: the probability density of GCP3–GFP intensities (yellow) fitted to a weighted sum of N -mers of GFP (dashed line) (a representative experiment). Weighted probability densities of individual GFP N -mer intensities (×1, ×2, …) are plotted beneath. See also Extended Data Fig. . b , A 3D reconstruction of γ-TuRC (12,851 particles) from negative-stain EM data and rigid body fit of repeating γ-tubulin/GCP2 subcomplexes (from PDB ID: 6V6S (ref. )) individually docked into the γ-TuRC density map. Fits for two subcomplexes at the γ-TuRC ‘seam’ were not reliable and are therefore omitted (Extended Data Fig. ). c , e – h , Left: maximum intensity projections of 10 min videos acquired after 20 min of incubation, showing microtubules (cyan) nucleated from γ-TuC (yellow) in the presence of either tubulin alone ( c ) or together with mCherry–EB3 ( e ) or mCherry–CDK5RAP2 ( f ) or mCherry–CLASP2 ( g ) or chTOG–mCherry ( h ) in the indicated conditions. In f and g , γ-TuC was also pre-incubated with indicated proteins (‘Premix’); experiments without pre-incubation are labelled as ‘No premix’. The arrowheads in insets indicate colocalizing particles. Right: representative kymographs and schemes illustrating microtubule dynamics and re-nucleation events (thin white arrows). Minus and plus indicate the two microtubule ends. The black arrowheads on top of kymographs indicate γ-TuC position and yellow and magenta arrows indicate γ-TuC and other proteins. The magnification is the same in c and e – h . d , Efficiency (mean ± s.e.m.) of microtubule nucleation by γ-TuC in the presence of either tubulin alone ( n = 3) or together with mCherry–EB3 ( n = 3), mCherry–CDK5RAP2 ( n = 3), mCherry–CDK5RAP2 pre-incubated with γ-TuC ( n = 3), mCherry–CLASP2 ( n = 4), mCherry–CLASP2 pre-incubated with γ-TuC ( n = 3), chTOG–mCherry ( n = 4) or chTOG–mCherry pre-incubated with γ-TuC (n = 3), where n is the number of independent experiments. ND, could not be determined. Representative images are shown on the left of c and e – h and Extended Data Fig. . Data points represent single fields of view for the given time point per experiment. Data points in cyan (0–10 min) were acquired from a smaller field of view; data points at 10–20 min and 20–30 min were acquired from a larger field of view shown in c and e – h .

Article Snippet: Membranes were blocked in 2% BSA in 0.02% Tween-20 in PBS for 30 min at room temperature followed by overnight incubation with primary antibodies (rabbit polyclonal anti-GFP (1:4,000, ab290, Abcam); mouse anti-GCP3 (1:1,000, sc-373758, Santa Cruz); mouse monoclonal anti-GCP6 (1:500, sc-374063, Santa Cruz), mouse monoclonal anti-GCP5 (1:500, sc-365837, Santa Cruz), mouse monoclonal anti-GCP2 (1:500, sc-377117, Santa Cruz), rabbit polyclonal anti-GCP4 (1:1,000, PA5-30557, Thermo Fisher), mouse monoclonal anti-γ-tubulin (1:10,000, T6557, GTU-88, Sigma), rabbit polyclonal anti-CDK5RAP2 (1:500, A300-554A, Bethyl Laboratories) and mouse monoclonal anti-Ku80 (1:2,000, 611360, BD Biosciences)) at 4 °C, followed by three washes with 0.02% Tween-20 in PBS, 1 h incubation with secondary antibodies (1:15,000, goat anti-rabbit IRDye-800CW (cat. no. 926-32211) and goat anti-mouse IRDye-680LT (cat. no. 926-68020) from Li-Cor Biosciences) at room temperature and a final three washes.

Techniques: Purification, Fluorescence, Staining, Incubation

Journal: Nature Cell Biology

Article Title: CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

doi: 10.1038/s41556-024-01366-2

Figure Lengend Snippet: a , Transmission EM (TEM) micrograph of negatively stained γ-TuRC. Inset shows 4X magnified view of a single γ-TuRC, scale bar-25nm. b , Two views of a 3D reconstruction of the γ-TuRC from negative-stain EM data. c , Rigid body fit of repeating γ-tubulin/GCP2 subcomplexes (from PDB ID: 6V6S ref. ) individually docked into the γ-TuRC density map. Fits for two subcomplexes at the γ-TuRC ‘seam’ were not reliable; and are therefore, omitted for clarity. d , Transmission EM (TEM) micrograph of negatively stained γ-TuRC prepared in complex with 120 nM CDK5RAP2. Inset shows 4X magnified view of a single γ-TuRC, scale bar-25nm. e , Two views of a 3D reconstruction of the γ-TuRC + CDK5RAP2 preparation from negative-stain EM data. f , Rigid body fit of repeating γ-tubulin/GCP2 subcomplexes (from PDB ID: 6V6S ref. ) individually docked into the γ-TuRC + CDK5RAP2 density map. As in c , fits for two subcomplexes at the γ-TuRC ‘seam’ were not reliable; and are therefore, omitted for clarity. g , Two views of the γ-tubulin rings from rigid body fitted models in c (γ-TuRC) and f (γ-TuRC + CDK5RAP2). h , Plots of the change in helical radius ( r) and helical pitch ( Z ) relative to β -tubulin in the 13-protofilament microtubule lattice (grey dashed line; PDB ID: 2HXF ref. and EMD- 5193 ref. ) calculated for γ-tubulin rings from γ-TuRC alone (orange), γ-TuRC + 120 nM CDK5RAP2 (green), and the γ-TuSC oligomer in the ‘closed’ state (blue; PDB ID: 5FLZ ref. ). See Methods for analysis details. i , Left: Processing workflow for generating a negative-stain EM 3D reconstruction of γ-TuRC. Right: Unmasked FSC curve for the γ-TuRC reconstruction. FSC = 0.5 is indicated by a dashed gray line, and an estimate of the corresponding resolution is indicated. j , Left: Processing workflow for generating a negative-stain EM 3D reconstruction of γ-TuRC in the presence of 120 nM CDK5RAP2. Right: Unmasked FSC curve for the γ-TuRC + CDK5RAP2 reconstruction. FSC = 0.5 is indicated by a dashed gray line, and an estimate of the corresponding resolution is indicated.

Article Snippet: Membranes were blocked in 2% BSA in 0.02% Tween-20 in PBS for 30 min at room temperature followed by overnight incubation with primary antibodies (rabbit polyclonal anti-GFP (1:4,000, ab290, Abcam); mouse anti-GCP3 (1:1,000, sc-373758, Santa Cruz); mouse monoclonal anti-GCP6 (1:500, sc-374063, Santa Cruz), mouse monoclonal anti-GCP5 (1:500, sc-365837, Santa Cruz), mouse monoclonal anti-GCP2 (1:500, sc-377117, Santa Cruz), rabbit polyclonal anti-GCP4 (1:1,000, PA5-30557, Thermo Fisher), mouse monoclonal anti-γ-tubulin (1:10,000, T6557, GTU-88, Sigma), rabbit polyclonal anti-CDK5RAP2 (1:500, A300-554A, Bethyl Laboratories) and mouse monoclonal anti-Ku80 (1:2,000, 611360, BD Biosciences)) at 4 °C, followed by three washes with 0.02% Tween-20 in PBS, 1 h incubation with secondary antibodies (1:15,000, goat anti-rabbit IRDye-800CW (cat. no. 926-32211) and goat anti-mouse IRDye-680LT (cat. no. 926-68020) from Li-Cor Biosciences) at room temperature and a final three washes.

Techniques: Transmission Assay, Staining

Journal: Nature Cell Biology

Article Title: CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

doi: 10.1038/s41556-024-01366-2

Figure Lengend Snippet: a,c,d,e , Left: maximum intensity projections and representative kymographs illustrating microtubule dynamics in 10 min time-lapse videos, acquired after 20 min of incubation, showing microtubules (cyan) nucleated from γ-TuC (GCP3-GFP, yellow) in the presence of 17.5 μM tubulin (17 μM unlabeled porcine tubulin and 0.5 μM HiLyte647-tubulin), 50 mM KCl and together with indicated concentrations of indicated proteins (magenta) and without any preincubation: 200 nM mCherry-EB3 ( a ); or 30 nM and 60 nM mCherry-CDK5RAP2 ( c ); or 30 nM and 200 nM mCherry-CLASP2 ( d ); or 50 nM chTOG-mCherry ( e ). Minus and plus represent the two microtubule ends. Black arrowheads on top of kymographs indicate γ-TuC position. Magenta arrows point to the signal of proteins, while yellow arrows point towards γ-TuC. Asterisks show rescues. Magnification for c-e is same as in a . Right: Quantification of average microtubule nucleation efficiency of γ-TuC as indicated: 200 nM mCherry-EB3 (n = 3); 30 nM (n = 3) or 60 nM (n = 3) mCherry-CDK5RAP2; 30 nM (n = 4) or 200 nM mCherry-CLASP2 (n = 3); 50 nM (n = 4) or 200 nM chTOG-mCherry (n = 4); where n is the number of independent experiments analyzed, also see Fig. . The plots present mean±s.e.m., and each data point represents a single field of view for the given time points per experiment. Data points at 0–10 min were acquired from a smaller field of view, whereas the data points at 10–20 min and 20–30 min acquired from a larger field of view (shown in the panels on the left). Data points for concentrations already shown in Fig. are from Fig. , replotted here for comparison. b , Purified mCherry-CDK5RAP2, mCherry-CLASP2 or chTOG-mCherry analyzed by Coomassie-stained SDS-PAGE.

Article Snippet: Membranes were blocked in 2% BSA in 0.02% Tween-20 in PBS for 30 min at room temperature followed by overnight incubation with primary antibodies (rabbit polyclonal anti-GFP (1:4,000, ab290, Abcam); mouse anti-GCP3 (1:1,000, sc-373758, Santa Cruz); mouse monoclonal anti-GCP6 (1:500, sc-374063, Santa Cruz), mouse monoclonal anti-GCP5 (1:500, sc-365837, Santa Cruz), mouse monoclonal anti-GCP2 (1:500, sc-377117, Santa Cruz), rabbit polyclonal anti-GCP4 (1:1,000, PA5-30557, Thermo Fisher), mouse monoclonal anti-γ-tubulin (1:10,000, T6557, GTU-88, Sigma), rabbit polyclonal anti-CDK5RAP2 (1:500, A300-554A, Bethyl Laboratories) and mouse monoclonal anti-Ku80 (1:2,000, 611360, BD Biosciences)) at 4 °C, followed by three washes with 0.02% Tween-20 in PBS, 1 h incubation with secondary antibodies (1:15,000, goat anti-rabbit IRDye-800CW (cat. no. 926-32211) and goat anti-mouse IRDye-680LT (cat. no. 926-68020) from Li-Cor Biosciences) at room temperature and a final three washes.

Techniques: Incubation, Comparison, Purification, Staining, SDS Page

Journal: Nature Cell Biology

Article Title: CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

doi: 10.1038/s41556-024-01366-2

Figure Lengend Snippet: a , b , Plus-end growth rate ( a ) and catastrophe frequency ( b ) of microtubules grown in indicated conditions, shown in Fig. and Extended Data Fig. with representative kymographs on the right. The numbers of the growth events analysed, n , from three independent experiments, are indicated. One-way analysis of variance (ANOVA) tests with Dunnett’s multiple comparisons corrected for multiple testing. c , Representative still images showing colocalization of the indicated premixed proteins. The enlargements show separate channels for γ-TuC/control (top) and NPFs (bottom). The arrows indicate colocalizing particles. d , e , Colocalization (mean ± s.e.m) of γ-TuC with indicated NPFs ( d ) and vice versa ( e ) under experimental conditions shown in c and in Fig. , and Extended Data Fig. . Data points represent the percentage of γ-TuCs colocalizing with the indicated protein or vice versa in n fields of view, in N independent experiments. The plots show data for γ-TuC premixed with mCherry without tubulin ( n = 63, N = 3), γ-TuC with mCherry–CDK5RAP2 and tubulin ( n = 9, N = 3), γ-TuC premixed with mCherry–CDK5RAP2 with ( n = 9, N = 3) and without tubulin ( n = 47, N = 3), γ-TuC with mCherry–CLASP2 and tubulin ( n = 4, N = 4), γ-TuC premixed with mCherry–CLASP2 with ( n = 3, N = 3) and without tubulin ( n = 48, N = 3), γ-TuC with chTOG–mCherry and tubulin ( n = 12, N = 4), γ-TuC premixed with chTOG–mCherry with ( n = 9, N = 3) and without tubulin ( n = 51, N = 3), GFP control premixed with mCherry without tubulin ( n = 80, N = 4), GFP control premixed with mCherry–CDK5RAP2 without tubulin ( n = 60, N = 3), GFP control premixed with mCherry–CLASP2 without tubulin ( n = 60, N = 3) and GFP control premixed with chTOG–mCherry without tubulin ( n = 59, N = 3). ** P = 0.0068, *** P = 0.0003 and **** P < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons corrected for multiple testing. f , Microtubule (MT) re-nucleation efficiency (mean ± s.e.m) from experiments shown in Fig. . Data points represent percentage of γ-TuCs re-nucleating microtubules in a single experiment. n , the number of independent experiments analysed and m , the number of γ-TuCs which nucleated microtubules that underwent depolymerization, pooled from all three 10 min videos of all experiments. Not significant (NS) P = 0.0704, *** P = 0.0007 and **** P < 0.0001, one-way ANOVA test with Tukey’s multiple comparisons corrected for multiple testing. ND, could not be determined.

Article Snippet: Membranes were blocked in 2% BSA in 0.02% Tween-20 in PBS for 30 min at room temperature followed by overnight incubation with primary antibodies (rabbit polyclonal anti-GFP (1:4,000, ab290, Abcam); mouse anti-GCP3 (1:1,000, sc-373758, Santa Cruz); mouse monoclonal anti-GCP6 (1:500, sc-374063, Santa Cruz), mouse monoclonal anti-GCP5 (1:500, sc-365837, Santa Cruz), mouse monoclonal anti-GCP2 (1:500, sc-377117, Santa Cruz), rabbit polyclonal anti-GCP4 (1:1,000, PA5-30557, Thermo Fisher), mouse monoclonal anti-γ-tubulin (1:10,000, T6557, GTU-88, Sigma), rabbit polyclonal anti-CDK5RAP2 (1:500, A300-554A, Bethyl Laboratories) and mouse monoclonal anti-Ku80 (1:2,000, 611360, BD Biosciences)) at 4 °C, followed by three washes with 0.02% Tween-20 in PBS, 1 h incubation with secondary antibodies (1:15,000, goat anti-rabbit IRDye-800CW (cat. no. 926-32211) and goat anti-mouse IRDye-680LT (cat. no. 926-68020) from Li-Cor Biosciences) at room temperature and a final three washes.

Techniques:

Journal: Nature Cell Biology

Article Title: CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

doi: 10.1038/s41556-024-01366-2

Figure Lengend Snippet: a , Maximum intensity projections of 10 min time-lapse videos, acquired after 20 min of incubation, showing microtubules nucleated from γ-TuC in the indicated conditions. b , The average microtubule nucleation efficiency (mean ± s.e.m.) from experiments shown in a . The numbers of fields of view analysed, n , from N independent experiments are indicated. Control data are from Fig. , replotted here for comparison, colours of the data points are the same as in Fig. . c – e , Still frames from 10 min videos (0:00 min is the starting point of the video) showing CAMSAP3 binding to the γ-TuC-anchored microtubule minus ends under the indicated experimental conditions. Left: microtubule release from γ-TuC (yellow arrows) colocalizing with CAMSAP3 (magenta arrows). Right: CAMSAP3 binding without microtubule release. The insets show cropped individual channels for γ-TuC (left) and CAMSAP3 (right), magnification is the same as merged images. Left: microtubule release from incomplete γ-TuRC (dim GFP signal) ( c ). Left: γ-TuC dissociation from minus end and also from glass surface within 6 min, followed by rescue at the microtubule plus end from CAMSAP3-stabilized stretch ( e ). f , The percentage (mean ± s.e.m) of γ-TuC-anchored microtubule minus ends colocalizing with CAMSAP3, from experiments represented in a and c – e . g , The percentage (mean ± s.e.m) of microtubules released from the γ-TuC colocalizing with CAMSAP3, shown in f . n , the number of independent experiments (plotted) and l , the total number of active γ-TuCs in f and the number of active γ-TuCs colocalized with CAMSAP3 in g , analysed over 10 min duration. Control data are from Fig. for f and from Fig. for g (GCP3 values), replotted here for comparison. One-way ANOVA with uncorrected Fisher’s LSD tests were used to compare the means with control. In b and f – h , control is black, CDK5RAP2 is red, CLASP2 is blue and chTOG is purple. h , The frequency of microtubule (MT) release from active γ-TuC in the presence of SNAP–AF647–CAMSAP3 under the experimental conditions shown in a and c – e . n , the numbers of active γ-TuCs analysed from N independent experiments. Data for GCP3 with or without CAMSAP3 are from Fig. , replotted here for comparison. CDK5RAP2 is abbreviated as C5R2 in the plots.

Article Snippet: Membranes were blocked in 2% BSA in 0.02% Tween-20 in PBS for 30 min at room temperature followed by overnight incubation with primary antibodies (rabbit polyclonal anti-GFP (1:4,000, ab290, Abcam); mouse anti-GCP3 (1:1,000, sc-373758, Santa Cruz); mouse monoclonal anti-GCP6 (1:500, sc-374063, Santa Cruz), mouse monoclonal anti-GCP5 (1:500, sc-365837, Santa Cruz), mouse monoclonal anti-GCP2 (1:500, sc-377117, Santa Cruz), rabbit polyclonal anti-GCP4 (1:1,000, PA5-30557, Thermo Fisher), mouse monoclonal anti-γ-tubulin (1:10,000, T6557, GTU-88, Sigma), rabbit polyclonal anti-CDK5RAP2 (1:500, A300-554A, Bethyl Laboratories) and mouse monoclonal anti-Ku80 (1:2,000, 611360, BD Biosciences)) at 4 °C, followed by three washes with 0.02% Tween-20 in PBS, 1 h incubation with secondary antibodies (1:15,000, goat anti-rabbit IRDye-800CW (cat. no. 926-32211) and goat anti-mouse IRDye-680LT (cat. no. 926-68020) from Li-Cor Biosciences) at room temperature and a final three washes.

Techniques: Incubation, Comparison, Binding Assay

Journal: Nature Cell Biology

Article Title: CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

doi: 10.1038/s41556-024-01366-2

Figure Lengend Snippet: a , Representative images of single molecules of the indicated purified proteins (GFP in dark green, GFP–EB3 in light green and GCP3–GFP in yellow) immobilized on coverslips with anti-GFP nanobody. b , Histograms of single-molecule fluorescence intensities shown for one experiment represented in a . n = 12,841 for GFP, n = 21,670 for GFP–EB3 and n = 16,420 for GCP3–GFP, where n is the number of molecules analysed. a and b are representative of six independent experiments that yielded similar results. c – e , Averaged histogram of weights of N -mers of GFP determined from the fitting to the intensities of GCP3–GFP puncta (fitting similar to shown in Fig. , right), showing the numbers of GFP molecules per GCP3–GFP puncta in control (black) ( c ) or in the presence of either mCherry–CDK5RAP2 (red) pre-incubated with γ-TuC ( d ) or mCherry–CLASP2 (blue) pre-incubated with γ-TuC ( e ). Tubulin and SNAP–AF647–CAMSAP3 in the experimental conditions identical to Fig. . For control, N = 6, n = 12,841 for GFP and n = 16,420 for GCP3–GFP; for premix CDK5RAP2, N = 3, n = 6,286 for GFP and n = 14,768 for GCP3–GFP; for premix CLASP2, N = 4, n = 8,530 for GFP and n = 17,023 for GCP3–GFP, where N is the number of independent experiments and n is the number of molecules analysed. The plots present mean ± s.d. f – h , Histograms of the number of GFP molecules per GCP3–GFP puncta that were active (nucleating microtubules (MTs)) ( f ), active and colocalizing with CAMSAP3 ( g ) or active and releasing microtubules upon CAMSAP3 binding to the minus end ( h ), determined from the plots and experiments shown in c – e . i – k , Histograms of the number of GFP molecules per GCP3–GFP puncta that were active (nucleating microtubules), colocalizing with CAMSAP3 and releasing microtubules in control ( i ), premix CDK5RAP2 ( j ) and premix CLASP2 ( k ). Values are replotted here from f – h . The colour code in plots c – k : control, black; CDK5RAP2, red and CLASP2, blue.

Article Snippet: Membranes were blocked in 2% BSA in 0.02% Tween-20 in PBS for 30 min at room temperature followed by overnight incubation with primary antibodies (rabbit polyclonal anti-GFP (1:4,000, ab290, Abcam); mouse anti-GCP3 (1:1,000, sc-373758, Santa Cruz); mouse monoclonal anti-GCP6 (1:500, sc-374063, Santa Cruz), mouse monoclonal anti-GCP5 (1:500, sc-365837, Santa Cruz), mouse monoclonal anti-GCP2 (1:500, sc-377117, Santa Cruz), rabbit polyclonal anti-GCP4 (1:1,000, PA5-30557, Thermo Fisher), mouse monoclonal anti-γ-tubulin (1:10,000, T6557, GTU-88, Sigma), rabbit polyclonal anti-CDK5RAP2 (1:500, A300-554A, Bethyl Laboratories) and mouse monoclonal anti-Ku80 (1:2,000, 611360, BD Biosciences)) at 4 °C, followed by three washes with 0.02% Tween-20 in PBS, 1 h incubation with secondary antibodies (1:15,000, goat anti-rabbit IRDye-800CW (cat. no. 926-32211) and goat anti-mouse IRDye-680LT (cat. no. 926-68020) from Li-Cor Biosciences) at room temperature and a final three washes.

Techniques: Purification, Fluorescence, Incubation, Binding Assay

Journal: Nature Cell Biology

Article Title: CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

doi: 10.1038/s41556-024-01366-2

Figure Lengend Snippet: a , c , e , Representative immunofluorescence images of the indicated RPE1 cell lines with or without stable overexpression of GFP–CDK5RAP2 and stained for CAMSAP2 or EB1, as indicated in untreated cells ( a ) and 1 min after nocodazole washout ( c and e ). The insets show cropped GFP–CDK5RAP2 channel in red, magnification is the same as merged images. Cells with clearly visible GFP signal at the centrosome (or the centrosome and the Golgi in the wild-type cells) were selected for the analysis in all three cell lines. b , The number of CAMSAP2 stretches per square micron area (mean ± s.e.m.) quantified from experiments represented in a and using values from Extended Data Fig. . d , EB1 mean intensity (mean ± s.e.m.) normalized to wild-type average quantified from experiments represented in c . f , The intensity ratio (mean ± s.e.m.) of CAMSAP2 over EB1 normalized to wild-type average quantified from experiments represented in e and using values from panel d and Extended Data Fig. . In all plots, the numbers of cells analysed per genotype, n , from three independent experiments are indicated, and one-way ANOVA test with Tukey’s multiple comparisons corrected for multiple testing was used. C5R2, CDK5RAP2; KO, knockout.

Article Snippet: Membranes were blocked in 2% BSA in 0.02% Tween-20 in PBS for 30 min at room temperature followed by overnight incubation with primary antibodies (rabbit polyclonal anti-GFP (1:4,000, ab290, Abcam); mouse anti-GCP3 (1:1,000, sc-373758, Santa Cruz); mouse monoclonal anti-GCP6 (1:500, sc-374063, Santa Cruz), mouse monoclonal anti-GCP5 (1:500, sc-365837, Santa Cruz), mouse monoclonal anti-GCP2 (1:500, sc-377117, Santa Cruz), rabbit polyclonal anti-GCP4 (1:1,000, PA5-30557, Thermo Fisher), mouse monoclonal anti-γ-tubulin (1:10,000, T6557, GTU-88, Sigma), rabbit polyclonal anti-CDK5RAP2 (1:500, A300-554A, Bethyl Laboratories) and mouse monoclonal anti-Ku80 (1:2,000, 611360, BD Biosciences)) at 4 °C, followed by three washes with 0.02% Tween-20 in PBS, 1 h incubation with secondary antibodies (1:15,000, goat anti-rabbit IRDye-800CW (cat. no. 926-32211) and goat anti-mouse IRDye-680LT (cat. no. 926-68020) from Li-Cor Biosciences) at room temperature and a final three washes.

Techniques: Immunofluorescence, Over Expression, Staining, Knock-Out

Journal: Nature Cell Biology

Article Title: CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

doi: 10.1038/s41556-024-01366-2

Figure Lengend Snippet: a , Western blot of cell lysates from wild type, AKAP450/CDK5RAP2/MMG knockouts, and wild type, AKAP450 knockouts and AKAP450/CDK5RAP2/MMG knockouts stably expressing GFP-CDK5RAP2, blotted using rabbit anti-CDK5RAP2 antibody (top) and mouse anti-Ku80 antibody (bottom). Top: red arrow indicates overexpressed GFP-CDK5RAP2 and black arrow indicates endogenous CDK5RAP2. GFP-CDK5RAP2 cell lines in the wild-type and AKAP450 background were clonal , whereas GFP-CDK5RAP2 triple CDK5RAP2/Myomegalin/AKAP450 knockout cells were a mixed cell population with respect to the GFP-CDK5RAP2 transgene. In the clonal lines, GFP-CDK5RAP2 overexpression was estimated to be 6–8-fold to the respective endogenous levels. See also the Methods section. b, c , Area of cells in square microns (mean±s.e.m.) ( b ) and number of CAMSAP2 stretches per cell (mean±s.e.m.) ( c ) quantified from experiments represented in Fig. . Number of cells analyzed, n, from three independent experiments in all conditions, are indicated. d , Representative immunofluorescence images of indicated cell lines with or without stable expression of GFP-CDK5RAP2 (red), stained for α-tubulin (cyan). Insets show cropped CDK5RAP2 channel in red, magnification is same as merged images. e,f , Total tubulin intensity per cell (mean±s.e.m.) normalized to wild-type average ( e ) and tubulin density per square micron area (mean±s.e.m.) normalized to wild-type average ( f ) quantified from experiments represented in d and Fig. and using values in panels b and e . Number of cells analyzed, n, from three independent experiments in all conditions, are indicated. g , CAMSAP2 mean intensity per cell (mean±s.e.m.) normalized to wild-type average quantified from experiments represented in Fig. . Number of cells analyzed, n, from three independent experiments in all conditions, are indicated. One-way ANOVA test with Tukey’s multiple comparisons corrected for multiple testing in b,c,e-g . ns, not significant. CDK5RAP2 is abbreviated as C5R2 in figure panels.

Article Snippet: Membranes were blocked in 2% BSA in 0.02% Tween-20 in PBS for 30 min at room temperature followed by overnight incubation with primary antibodies (rabbit polyclonal anti-GFP (1:4,000, ab290, Abcam); mouse anti-GCP3 (1:1,000, sc-373758, Santa Cruz); mouse monoclonal anti-GCP6 (1:500, sc-374063, Santa Cruz), mouse monoclonal anti-GCP5 (1:500, sc-365837, Santa Cruz), mouse monoclonal anti-GCP2 (1:500, sc-377117, Santa Cruz), rabbit polyclonal anti-GCP4 (1:1,000, PA5-30557, Thermo Fisher), mouse monoclonal anti-γ-tubulin (1:10,000, T6557, GTU-88, Sigma), rabbit polyclonal anti-CDK5RAP2 (1:500, A300-554A, Bethyl Laboratories) and mouse monoclonal anti-Ku80 (1:2,000, 611360, BD Biosciences)) at 4 °C, followed by three washes with 0.02% Tween-20 in PBS, 1 h incubation with secondary antibodies (1:15,000, goat anti-rabbit IRDye-800CW (cat. no. 926-32211) and goat anti-mouse IRDye-680LT (cat. no. 926-68020) from Li-Cor Biosciences) at room temperature and a final three washes.

Techniques: Western Blot, Stable Transfection, Expressing, Knock-Out, Over Expression, Immunofluorescence, Staining

Journal: Nature Cell Biology

Article Title: CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

doi: 10.1038/s41556-024-01366-2

Figure Lengend Snippet: a , A scheme of the γ-TuRC capping assays. b , Maximum intensity projections of 5 min videos showing γ-TuC capping of GMPCPP-stabilized microtubules (MTs), in the presence of tubulin with (right) or without (left) mCherry–CDK5RAP2. The enlarged views show capped (top) and uncapped minus ends (bottom), distinguished from the plus ends by the absence of growth or slow growth dynamics. The yellow arrowheads and inset (scale bar, 1 μm) at the right bottom shows colocalization of γ-TuC with CDK5RAP2. c , Minus-end capping efficiency (mean ± s.e.m.) of γ-TuC for stabilized microtubules with different protofilament numbers in absence or presence of mCherry–CDK5RAP2 from experiments represented in a and b . n , number of fields of view analysed (plotted) and m , number of microtubule minus ends analysed from N independent experiments, as indicated. NS, one-way ANOVA test with Šídák’s multiple comparisons corrected for multiple testing. d , e , Single frames and representative kymographs from 10 min videos, showing microtubules growing from GMPCPP-stabilized microtubule seeds (short green lines below kymographs) in the presence of either mCherry–CDK5RAP2 and tubulin only ( d ) or together with GFP–EB3 ( e ) in the indicated conditions. Fluorescent tubulin was substituted with unlabelled tubulin in the assays with GFP–EB3. The arrowheads show CDK5RAP2 binding to microtubule minus ends, arrows show binding to plus ends and asterisks show binding to microtubule lattice. Minus and plus represent the two microtubule ends. The magnification is the same in d and e . f , A model of microtubule NPFs, CDK5RAP2 and CLASP2, which can activate full and partial γ-TuRCs to nucleate a microtubule. The minus end of such microtubules may or may not be fully anchored to γ-TuC allowing some protofilaments that are not attached to the γ-tubulin subunits to attain a flared conformation permissive for CAMSAP binding. CAMSAP2 or 3 bind to the minus end of the γ-TuRC-capped microtubule at an intradimer site between two protofilaments , where they can promote minus-end polymerization, stabilize the growing minus end or alter lattice conformation. Elongating protofilaments at the minus end can generate a pushing force or can alter the conformation of the microtubule lattice , , causing detachment of neighbouring protofilaments from γ-TuRC and microtubule release. CDK5RAP2 inhibits microtubule release by suppressing CAMSAP binding, probably by modifying the γ-TuRC conformation and/or γ-TuRC–microtubule interface.

Article Snippet: Membranes were blocked in 2% BSA in 0.02% Tween-20 in PBS for 30 min at room temperature followed by overnight incubation with primary antibodies (rabbit polyclonal anti-GFP (1:4,000, ab290, Abcam); mouse anti-GCP3 (1:1,000, sc-373758, Santa Cruz); mouse monoclonal anti-GCP6 (1:500, sc-374063, Santa Cruz), mouse monoclonal anti-GCP5 (1:500, sc-365837, Santa Cruz), mouse monoclonal anti-GCP2 (1:500, sc-377117, Santa Cruz), rabbit polyclonal anti-GCP4 (1:1,000, PA5-30557, Thermo Fisher), mouse monoclonal anti-γ-tubulin (1:10,000, T6557, GTU-88, Sigma), rabbit polyclonal anti-CDK5RAP2 (1:500, A300-554A, Bethyl Laboratories) and mouse monoclonal anti-Ku80 (1:2,000, 611360, BD Biosciences)) at 4 °C, followed by three washes with 0.02% Tween-20 in PBS, 1 h incubation with secondary antibodies (1:15,000, goat anti-rabbit IRDye-800CW (cat. no. 926-32211) and goat anti-mouse IRDye-680LT (cat. no. 926-68020) from Li-Cor Biosciences) at room temperature and a final three washes.

Techniques: Binding Assay

Journal: Current Biology

Article Title: Microtubule nucleation from the fibrous corona by LIC1-pericentrin promotes chromosome congression

doi: 10.1016/j.cub.2023.01.010

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CDK5RAP2 , Bethyl , Cat# A300-554A; RRID: AB_477974.

Techniques: Recombinant, Sequencing, Expressing, Plasmid Preparation, Software, Imaging

Journal: IBRO Neuroscience Reports

Article Title: An alternative splice isoform of mouse CDK5RAP2 induced cytoplasmic microtubule nucleation

doi: 10.1016/j.ibneur.2022.09.004

Figure Lengend Snippet: An alternative splice isoform of CDK5RAP2. (A) mRNA structure of mouse CDK5RAP2. CM1 domain refers to the centrosomin N-terminal motif 1, which binds to and activates γTuRC. The C-terminal domain is responsible for the localization of CDK5RAP2 at the centrosome and at the Golgi apparatus. Primers used for RT-PCR were shown. (B) RT-PCR analysis on the exon 17 skipping. Total RNA from embryonic day 13.5 brain, postnatal day 1 (P1D), and postnatal day 7 cerebrum were analyzed with primers flanking exon 17 (Pr1 and Pr5). Skipping of exon 17 (Δe17) was detected after P1D. (C) Amplicons with primer sets used for real time-RT-PCR shown in (D). DNAs were eluted from the upper and the lower bands of the gel shown in (B) and were amplified with primers specific to each isoform. (D) Quantification of the expression of CDK5RAP2 isoforms measured with real-time RT-PCR. The full-length isoform was amplified with primer set Pr2 and Pr4, and the Δe17 isoform was amplified with primer set Pr3 and Pr4. Error bars are S. E. (E) Real-time PCR analysis on the change in the isoform expression during the culture of primary cortical neurons. DIV stands for days in vitro. (F) Detection of CDK5RAP2 proteins in tissues. Lysates from the P7D cerebrum and P21D testis were immunoprecipitated with anti-CDK5RAP2 antibody and immunoblotted with the same antibody. The star indicates the band with a molecular weight close to the calculated molecular weight of Δe17 isoform.

Article Snippet: Antibodies and their dilutions were as follows: rabbit polyclonal antibody against pericentrin (Covance #923701, Burlington, NC) [1:500], rabbit polyclonal antibody against CDK5RAP2 (MILLIPORE #06–1398, Burlington, MA) [1:500], mouse monoclonal antibody against α-tubulin (Sigma-Aldrich #T5168, St. Louis, MO) [1:500], rabbit polyclonal antibody against α-tubulin (Thermo Fisher Scientific #RB-9281, Waltham, MA) [1:200], mouse monoclonal antibody against acetylated tubulin (Sigma-Aldrich #T6793) [1:500], goat polyclonal antibody against γ-tubulin (Santa Cruz #sc-7396, Santa Cruz, CA) [1:500], mouse monoclonal antibody against FLAG (clone M2; Sigma-Aldrich) [1:1000] and mouse monoclonal antibody against GM130 (BD Biosciences #610822, Franklin Lakes, NJ) [1:400].

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Expressing, Real-time Polymerase Chain Reaction, In Vitro, Immunoprecipitation, Molecular Weight

Journal: IBRO Neuroscience Reports

Article Title: An alternative splice isoform of mouse CDK5RAP2 induced cytoplasmic microtubule nucleation

doi: 10.1016/j.ibneur.2022.09.004

Figure Lengend Snippet: Δe17 isoform localized diffusely in cytoplasm and binds to γTuRC in HEK293T cells. Cells were observed with a laser confocal microscope. (A) Immunostaining with anti-CDK5RAP2 antibody and GM130 antibody (Golgi marker). Endogenous CDK5RAP2 localized intensely at the centrosome (arrow) and weakly on the Golgi apparatus. (B) Anti-FLAG immunostaining of FLAG-tagged full-length CDK5RAP2. The full-length isoform localized intensely at the centrosome (arrow) and weakly on the Golgi apparatus. (C) Localization of FLAG-tagged Δe17 isoform in cells expressing low level of the isoform. FLAG immunostaining showed diffuse localization of the isoform in the cytoplasm as well as weak localization at the centrosome (arrow). (D) Localization of FLAG-tagged Δe17 isoform in cells expressing low level of the isoform. FLAG immunostaining did not show obvious localization of the isoform at the Golgi apparatus (arrow). (E) Localization of FLAG-tagged Δe17 isoform in cells expressing a high level of the isoform. FLAG immunostaining showed diffuse and even localization in the cytoplasm. (F) Interaction of Δe17 isoform with γTuRC. Lysates of cells transfected with FLAG-tagged CDK5RAP2 isoforms were immunoprecipitated with FLAG antibody and were immunoblotted with anti-γ-tubulin antibody and with anti-MZT2 antibody.

Article Snippet: Antibodies and their dilutions were as follows: rabbit polyclonal antibody against pericentrin (Covance #923701, Burlington, NC) [1:500], rabbit polyclonal antibody against CDK5RAP2 (MILLIPORE #06–1398, Burlington, MA) [1:500], mouse monoclonal antibody against α-tubulin (Sigma-Aldrich #T5168, St. Louis, MO) [1:500], rabbit polyclonal antibody against α-tubulin (Thermo Fisher Scientific #RB-9281, Waltham, MA) [1:200], mouse monoclonal antibody against acetylated tubulin (Sigma-Aldrich #T6793) [1:500], goat polyclonal antibody against γ-tubulin (Santa Cruz #sc-7396, Santa Cruz, CA) [1:500], mouse monoclonal antibody against FLAG (clone M2; Sigma-Aldrich) [1:1000] and mouse monoclonal antibody against GM130 (BD Biosciences #610822, Franklin Lakes, NJ) [1:400].

Techniques: Microscopy, Immunostaining, Marker, Expressing, Transfection, Immunoprecipitation